Structural basis of membrane targeting and regulation of the innate immunity adaptor TIRAP by its phosphoinositide-binding motif

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Toll-like receptors (TLRs) are the main components of the innate immunity. Pathogen-activated TLRs trigger a cytoplasmic signaling cascade through adaptor proteins, with the first being the TIR domain-containing adaptor protein (TIRAP). TIRAP contains a TIR domain, which associates with TLRs and other adaptor proteins; and a N-terminal phosphoinositide-binding motif (PBM) that mediates the membrane recruitment of TIRAP. Upon ligand activation, TLRs are recruited to the phosphoinositide (PIP)-enriched region in the membrane, where TIRAP recruits other adaptors to the membrane to activate TLR signaling pathway. To investigate the mechanism of membrane targeting of TIRAP and the basis for its regulation, I functionally and structurally characterized TIRAP and its PBM using biophysical approaches. I show that TIRAP PBM adopts helical structural in dodecylphosphocholine (DPC) micelles and other membrane mimics. NMR studies reveal that TIRAP PBM binds PIPs following a fast exchange regime with a moderate affinity through two conserved basic termini. Mutation of these two basic regions abolishes PIPs binding without distorting the helical structure of the peptide. Solution NMR structure of TIRAP PBM exhibits a central relatively hydrophobic helix surrounded by the flexible N- and C-termini. Paramagnetic studies indicate that the helix is close to the micelle core, whereas two termini are located on the micellar surface. Nuclear spin relaxation experiments indicate that the two termini of TIRAP PBM become more ordered when bound to PIP, thus, we propose that the central helix in PBM is responsible for membrane insertion, whereas the two sets of basic residues interact with PIPs to stabilize TIRAP's membrane interaction. Phosphomimetic mutation of Thr28 to Asp (T28D) as well as phosphorylation in Thr28 inhibit TIRAP PBM's binding to phosphoinositides by distorting the central helical structure of the peptide. More importantly, TIRAP T28D disrupt its subcellular localization in vivo. Thus, phosphorylation can impair proper insertion of TIRAP at the plasma membrane through PBM and, consequently, it may represent the first signal that promotes TIRAP degradation.